PROJECT SUMMARY/ABSTRACT OF FUNDED AWARD NIH GM112735, Mechanisms of Spliceosome Assembly and Splice Site Selection RNA splicing?the removal of introns and ligation of exons?is an essential step in eukaryotic gene expression and must occur precisely. Precision depends on accurate recognition of the splice sites within RNAs by a macromolecular machine called the spliceosome. Spliceosomes are assembled at particular locations in transcripts from protein and small nuclear ribonucleoprotein (snRNP) components. In humans, most RNAs are alternatively spliced meaning that the spliceosome can incorporate multiple regulatory signals to control the splicing fate of a given transcript. Many of the key steps in regulating alternative splicing and splicing efficiency occur in the earliest stages of spliceosome assembly. During these steps in yeast, the 5' splice site (SS) and the branchsite (BS) are first recognized by the U1 snRNP and the BBP/Mud2 protein heterodimer, respectively. This forms the so-called commitment complex (CC) that then recruits U2 to form the pre- spliceosome. Pre-spliceosome formation is believed to determine the alternative splicing fate of many transcripts and defects in human CC and pre-spliceosome components are linked to genetic diseases including myelodysplastic syndrome (MDS). The ultimate goals of this project are to understand the pathways by which spliceosomes assemble on RNAs. While the identities of the players in these processes are known, their mechanisms of action remain unclear. Investigation of these events will lead to a better understanding of this fundamental process as well as provide new insights into diseases linked to splicing. Here, we focus on formation of the spliceosomal CC and its transition into the pre-spliceosome. In these experiments, we exploit the unique capabilities of single molecule fluorescence as our primary tool. In Aim 1, we will purify the components of CC and reconstitute its assembly in vitro. A key outcome of Aim 1 is a purified, biochemically characterized system for studying CC formation. This is a necessary step in our long-term objective of biochemically reconstituting spliceosome assembly. In Aim 2, we study the disassembly of single molecules of CC and the formation of pre-spliceosomes using a novel combination of purified components and yeast cell extracts. In Aim 3, we use a variety of approaches to study the binding and conformational dynamics of the Prp5 ATPase during pre- spliceosome formation. This administrative supplement will allow us to construct a powerful single molecule fluorescence microscope (CoSMoS 2.0) for furthering this research. It will greatly increase our temporal resolution, providing novel insights into CC interactions in Aims 1 and 2. CoSMoS 2.0 will also enhance Aim 3 by letting us probe ATP hydrolysis by Prp5 in real-time. These experiments will ultimately be critical for connecting ATP hydrolysis to discrete steps in splicing. Together our experiments will provide much needed new insights into spliceosome assembly and the ways in which it can be regulated.